Olivocochlear nicotinic synapses in the inner ear play a critical role in normal hearing. Olivocochlear pathway fibers originate in the brain and directly innervate mechanosensory hair cells. These synapses are required for normal hearing sensitivity, frequency selectivity and tonotopic map formation during development. However, little is known about the molecular mechanisms required for the proper assembly and function of Olivocochlear synapses. a9/10-nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in hair cells. Functional coupling of a9/10-nAChRs to small conductance Ca2+activated potassium (SK2) channels is essential for normal function. The objective of the proposed studies is to identify proteins required for synaptic localization and functional coupling of a9/10-nAChRs and SK2 channels. The proposed studies will: 1) define the molecular composition of Olivocochlear postsynaptic sites in hair cells, 2) test our hypothesis that specific adapter proteins bind to a9/10-nAChRs and SK2 channels and tether them to the postsynaptic scaffold, and 3) test the in vivo roles of the adapter proteins and the scaffold protein adenomatous polyposis coli (APC) in regulating the synaptic localization of a9/10-nAChRs and SK2. In Aim 1 studies, immunofluorescence and confocal microscopy will be used to identify protein components of the avian hair cell postsynaptic complex. In vitro and in vivo co-precipitation assays will be used to identify binding partners of a10 and SK2 that connect a9/10-nAChRs and SK2 channels to postsynaptic complex components. Aim 2 studies will test the roles of the adapter proteins and APC in directing the synaptic localization and functional coupling of a9/10-nAChRs and SK2 channels in vivo. We will use retroviral-mediated expression of dominant negative peptides in vivo that selectively block the targeted protein interactions: We will test for changes in the synaptic localization of a9/10-nAChRs and SK2 in hair cells expressing the dominant negative versus control peptides using immunofluorescence and quantitative confocal microscopy. Changes in functional coupling will be assayed using whole-cell patch-clamp recordings. Overall, these studies will provide novel insights into molecular mechanisms that direct functional Olivocochlear synapse assembly in sensory hair cells. Elucidating these mechanisms in normal developing hair cells will be essential for developing strategies to promote synapse formation in regenerating hair cells and restore hearing in animal models of sensorineural hearing loss, a permanent condition that affects millions of Americans.